WO2024037322A1 - HÉTÉRODIMÈRE FC/IL-15Rα DE SOUS UNITÉ FC PORTANT LA MUTATION IL-15 ET SON UTILISATION - Google Patents

HÉTÉRODIMÈRE FC/IL-15Rα DE SOUS UNITÉ FC PORTANT LA MUTATION IL-15 ET SON UTILISATION Download PDF

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WO2024037322A1
WO2024037322A1 PCT/CN2023/110205 CN2023110205W WO2024037322A1 WO 2024037322 A1 WO2024037322 A1 WO 2024037322A1 CN 2023110205 W CN2023110205 W CN 2023110205W WO 2024037322 A1 WO2024037322 A1 WO 2024037322A1
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protein
seq
mutant
cells
amino acid
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Chinese (zh)
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周海平
洪海燕
张雪亮
李重阳
谭晓璐
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海徕科(北京)生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2086—IL-13 to IL-16
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/91—Cell lines ; Processes using cell lines

Definitions

  • the invention belongs to the technical field of medical preparations, and specifically relates to IL-15 mutant-Fc/IL-15R ⁇ subunit-Fc heterodimer and its use.
  • IL-15 Human interleukin-15
  • T cells B cells and NK cells
  • IL-15 can activate, maintain and expand CD8+ memory T cells without activating regulatory T lymphocytes (Tregs, which have immunosuppressive functions).
  • IL-2 is the first cytokine to be identified. It was originally discovered from the culture supernatant of activated human T cells and is a soluble factor that mediates T cell proliferation. IL-2 was also the first cytokine approved by the FDA for cancer treatment. Although mainly secreted by CD4+ and CD8+ T cells under antigen stimulation, activated dendritic cells (DC), mast cells and NKT cells also produce small amounts of IL-2.
  • IL-15 has some similar functions to IL-2, including stimulating the proliferation of activated T cells, generating cytotoxic effector T cells, and activating and maintaining NK cells. They also promote B cells to induce immunoglobulin synthesis and regulate lymphatic homeostasis. But unlike IL-2, IL-15mRNA is expressed in various tissues, including hematopoietic cells and non-hematopoietic cells, such as keratinocytes, nerve cells, stromal cells, fibroblasts, etc. But unlike the widespread IL-15 mRNA expression, mature IL-15 protein production is mainly restricted to DCs and monocytes/macrophages.
  • IL-15 has a chemotactic effect on T cells: circulating lymphocytes home to peripheral lymph nodes, inhibit lymphocyte apoptosis, promote the activation and proliferation of T cells, and induce the production of cytotoxic T lymphocytes (CTL).
  • CTL cytotoxic T lymphocytes
  • IL-15 not only promotes the production of memory CD8+ T cells, but also plays a crucial role in maintaining the number of memory CD8+ T cells in the body.
  • NK cells IL-15 also plays an important role in their activation and proliferation. In mice overexpressing IL-15, the number of NK cells increased significantly and enhanced the immune response.
  • IL-15 also plays an important role in the functional maturation of DC cells and macrophages. In DC cells, IL-15 can promote DC cells to express costimulatory factors and IFN- ⁇ , and improve the ability of DC cells to activate CD8+ T cells and NK cells.
  • IL-2 and IL-15 are type I four-alpha helix bundle cytokines and belong to the gamma receptor cytokine family. This group of cytokines shares the same receptor subunit ⁇ c and exhibits pleiotropic effects in modulating innate and adaptive immune responses.
  • the receptors for IL-2 and IL-15 are heterotrimers, except for the cytokine receptor subunit ⁇ c (also known as IL-2R ⁇ or CD132); they also share a ⁇ subunit, here referred to as IL- 2/15R ⁇ (also known as CD122).
  • the third and unique receptor subunits of IL-2 and IL-15 are IL-2R ⁇ and IL-15R ⁇ respectively.
  • IL-2R ⁇ IL-2R ⁇
  • sIL-2R ⁇ soluble form
  • IL-15R ⁇ as a unique component of the IL-15 receptor complex, is mainly expressed on monocytes and dendritic cells. Different from other ⁇ c family cytokines (such as IL-2), IL-15 as a cytokine first binds to IL-15R ⁇ expressing cells, and then the IL-15/IL-15R ⁇ complex is presented to activated T cells or IL-2/15R ⁇ and ⁇ c on NK cells form a high-affinity immune synapse.
  • IL-15 as a cytokine first binds to IL-15R ⁇ expressing cells, and then the IL-15/IL-15R ⁇ complex is presented to activated T cells or IL-2/15R ⁇ and ⁇ c on NK cells form a high-affinity immune synapse.
  • IL-2/15R ⁇ Due to the shared receptor subunit (IL-2/15R ⁇ ), IL-2 and IL-15 trigger several similar downstream signaling pathways, including Janus kinases (JAKs) signal transducers and activators of transcription (STATs), JAK1 interacts with IL-2/15R ⁇ , and JAK3 interacts with ⁇ c.
  • JAKs Janus kinases
  • STATs Janus kinases
  • JAK1 interacts with IL-2/15R ⁇
  • JAK3 interacts with ⁇ c.
  • IL-2 and IL-15 also display distinct functions in the body, particularly in adaptive immune responses.
  • IL-2 is required for the development and maintenance of regulatory T cells (Tregs), and IL-2 is closely associated with activation-induced cell death (AICD).
  • AICD activation-induced cell death
  • IL-2 When IL-2 is used to treat tumors, it may cause capillary leak syndrome as a side effect.
  • IL-15 does not mediate AICD, but inhibits IL-2-induced AICD, and does not cause capillary leak syndrome side effects.
  • IL-15 is a major force supporting the persistence of natural killer (NK) cells and memory CD8+ T cells.
  • N-803 (Anktiva, ALT-803) is composed of an IL-15 mutant combined with the sushi region/IgG1Fc fusion protein of IL-15/IL-15R ⁇ . Compared with wild-type IL-15, N-803 has good pharmacokinetic properties, lasts longer in the body, and has stronger anti-tumor activity.
  • the technical problem to be solved by the present invention is how to provide better IL-15 drugs.
  • the present invention provides a heterodimeric protein, which includes protein a and protein b, where the protein a includes an IL-15 mutant and a first Fc mutant;
  • the protein b includes the IL-15R ⁇ subunit sushi domain and other partial fragments of the IL-15R ⁇ subunit and a second Fc mutant;
  • the first Fc mutant and the second Fc mutant are selected from Knob modified Fc or Hole modified Fc, and the modification type of Knob modification or Hole modification of the first Fc mutant and the second Fc mutant is different;
  • the IL-15 mutant has an amino acid sequence selected from positions 1-111 of SEQ ID No. 18, positions 1-111 of SEQ ID No. 15, positions 1-114 of SEQ ID No. 14, and SEQ ID No. 17 The polypeptide of any one of positions 1-114 of SEQ ID No. 16 and positions 1-114 of SEQ ID No. 13;
  • the IL-15R ⁇ subunit sushi domain and other partial fragments of the IL-15R ⁇ subunit are polypeptides whose amino acid sequence is SEQ ID No. 2.
  • the "sushi region" in the "IL-15R ⁇ subunit sushi domain and other partial fragments of IL-15R ⁇ subunit” described in the present invention mainly refers to the peptide segment whose amino acid sequence is SEQ ID No. 2 No. 1-65.
  • the "other partial fragments" in the "IL-15R ⁇ subunit sushi domain and other partial fragments of IL-15R ⁇ subunit” mentioned in the present invention mainly refer to the amino acid sequence of SEQ ID No. 2 No. 66-78 (DPALVHQRPAPPS) of peptides.
  • the protein a further includes a linker, which connects the IL-15 mutant and the first Fc mutant.
  • the linker can be (GGGGS)n, and n is a natural number from 0 to 4. In one embodiment of the present invention, n is 3.
  • the protein a includes: the IL-15 mutant, a linker connected to the C-terminal of the IL-15 mutant, and a first Fc mutant connected to the C-terminal of the linker;
  • the protein b includes: the IL-15R ⁇ subunit sushi domain and other partial fragments, and a second Fc mutant connected to the C-terminus of the IL-15R ⁇ subunit sushi domain and other partial fragments.
  • the first Fc mutant or the second Fc mutant may be the Fc of antibody IgG1, IgG2, IgG3 or IgG4 or a mutant thereof;
  • the first Fc mutant or the second Fc mutant is selected from Fc of IgG4 or a mutant thereof.
  • serine 228 in the hinge region can be mutated to proline to obtain a stable IgG4 Fc mutant.
  • the protein a and the protein b are formed by combining the Knob into hole of Fc.
  • the Fc domain of one chain contains T366W mutations
  • the corresponding domain of the other chain contains T366S, L368A, and Y407V mutations
  • the Fc domain of one chain contains T350V, L351Y, F405A, and Y407V mutations.
  • the corresponding domain of the other chain contains T350V, T360L, K392L and T394V mutations; or other forms of Knob into hole combinations.
  • the amino acid sequence of the knob-modified Fc (second Fc mutant) is SEQ ID No. 9, and its nucleotide sequence is SEQ ID No. 37; the hole-modified Fc The amino acid sequence of (the first Fc mutant) is SEQ ID No. 10, and its nucleotide sequence is SEQ ID No. 38.
  • the protein a can be any one of A1)-A7):
  • amino acid sequence is the protein of SEQ ID No. 18;
  • amino acid sequence is the protein of SEQ ID No. 15;
  • amino acid sequence is the protein of SEQ ID No. 14;
  • amino acid sequence is the protein of SEQ ID No. 17;
  • amino acid sequence is the protein of SEQ ID No. 16;
  • amino acid sequence is the protein of SEQ ID No. 13;
  • a fusion protein obtained by connecting a protein tag to the N-terminus or/and C-terminus of the protein described in any one of A1) to A6).
  • the protein b can be any one of A8)-A10):
  • amino acid sequence is the protein of SEQ ID No. 19;
  • amino acid sequence is the protein of SEQ ID No. 20;
  • a fusion protein obtained by connecting a protein tag to the N-terminus or/and C-terminus of the protein described in A8) or A9).
  • protein a in the heterodimeric protein is a protein whose amino acid sequence is SEQ ID No. 18, and protein b is a protein whose amino acid sequence is SEQ ID No. 19.
  • the present invention provides biological materials related to the above-mentioned heterodimeric proteins, and the biological materials are any of the following:
  • B2 An expression cassette containing the nucleic acid molecule described in B1);
  • B3 A recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2);
  • B4 A recombinant microorganism containing the nucleic acid molecule described in B1) or containing the expression cassette described in B2) or containing the recombinant vector described in B3);
  • B5 An animal cell line containing the nucleic acid molecule described in B1) or containing the expression cassette described in B2) or containing the recombinant vector described in B3);
  • B6 A plant cell line containing the nucleic acid molecule described in B1) or containing the expression cassette described in B2) or containing the recombinant vector described in B3);
  • the present invention provides a product, the active ingredient of the product is the above-mentioned heterodimeric protein; the use of the product is any of the following C1)-C6):
  • NK cells Improve the killing ability of NK cells against tumor cells (such as K562 cells).
  • the present invention provides a pharmaceutical composition, which is composed of the above-mentioned heterodimeric protein and other drugs.
  • the other drugs may be at least one of the following drugs: immune checkpoint drugs, cell engager bispecific antibodies, and cell therapy products.
  • the present invention provides the application of the above-mentioned heterodimeric protein or the above-mentioned biological material or the above-mentioned product or the above-mentioned pharmaceutical composition in any one of the following D1)-D12):
  • NK cells Improve the killing ability of NK cells against tumor cells (such as K562 cells).
  • the T cells include CD8+ T cells and/or CD4+ T cells.
  • the body may be a mammalian body.
  • the mammals include humans and mice.
  • the present invention provides a method for treating diseases, which method includes the following steps: administering the above-mentioned heterodimeric protein or the above-mentioned biological material or the above-mentioned product or the above-mentioned pharmaceutical combination to the patient. substance so that the patient can be treated.
  • the diseases include infectious diseases, tumors, blood diseases, inflammatory diseases and autoimmune diseases.
  • infectious diseases include but are not limited to viral infections (such as smallpox virus infection, HIV infection, HBV infection, etc.), bacterial infections, and fungal infections.
  • the tumors include, but are not limited to, melanoma, colorectal cancer, skin cancer, lymphoma, renal cell carcinoma, liver cancer, lung cancer, gastric cancer, and breast cancer.
  • the hematological diseases include, but are not limited to, anemia, acute myeloid leukemia, myelodysplastic syndrome, and T-cell large granular lymphocytic leukemia.
  • the autoimmune diseases include, but are not limited to, multiple sclerosis, psoriasis, rheumatoid arthritis, gastritis, and mucositis.
  • the product may be a drug or preparation.
  • carrier materials may also be added.
  • the carrier materials include but are not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carriers Materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.).
  • water-soluble carrier materials such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.
  • poorly soluble carrier materials such as ethyl cellulose, cholesterol stearate, etc.
  • enteric carriers Materials such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.
  • These materials can be used to make a variety of dosage forms, including but not limited to tablets, capsules, dropping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, Oral tablets, suppositories, freeze-dried powder injections, etc. It can be ordinary preparations, sustained-release preparations, controlled-release preparations and various particulate drug delivery systems.
  • carriers are, for example, diluents and absorbing agents such as starch, dextrin, calcium sulfate, lactose, glycerin, etc.
  • Alcohol sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and adhesives, such as water, glycerin, polyethylene glycol, ethanol, propanol, Starch slurry, dextrin, syrup, honey, glucose solution, arabic slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrating agents, such as dry starch , alginate, agar powder, fucoidyl starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium lauryl sulfonate, methyl cellulose, ethyl cellulose etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers, such as quaternary ammonium salts, sodium
  • Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bi-layer and multi-layer tablets.
  • a wide variety of carriers known in the art may be used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, and ethanol.
  • agar powder such as honey, liquid sugar, rice cereal or batter, etc.
  • disintegrating agents such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.
  • a wide variety of carriers known in the art may be used.
  • the carrier include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, and the like.
  • diluents commonly used in this field can be used, for example, water, ethanol, polyethylene glycol, 1, 3-Propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc.
  • an appropriate amount of sodium chloride, glucose or glycerin can be added to the injection preparation.
  • conventional co-solvents, buffers, pH adjusters, etc. can also be added.
  • colorants, preservatives, fragrances, flavoring agents, sweeteners or other materials can also be added to the pharmaceutical preparations.
  • the above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection.
  • the present invention invented an innovative IL-15 mutant-Fc/IL-15R ⁇ -Fc heterodimer protein, which has more biological activity than existing similar products.
  • the dimeric protein HL-015-9 provided by the present invention.
  • This dimeric protein can well stimulate the proliferation of NK cells and T cells, and can be used alone or in combination with other drugs (such as immune checkpoint drugs, cell engager bispecific antibodies or cell therapy products) to achieve Better results.
  • the heterodimeric protein provided by the present invention can improve the proliferation activity of CTLL-2 and Mo7e cells.
  • the heterodimeric protein provided by the present invention can improve the proliferation activity of NK cells, CD8+T cells, and CD4+T cells.
  • the heterodimeric protein provided by the present invention can promote NK cells to kill K562.
  • the dimeric protein HL-015-9 provided by the present invention inhibits tumor growth better than ALT-803 at the same injection dose.
  • Figure 1 shows the effects of different molecules designed in the first round on Mo7e cell proliferation.
  • Figure 2 shows the effects of different molecules designed in the first round on CTLL-2 cell proliferation.
  • Figure 3 shows the effect of the second round of designed IL-15 mutant-Fc/IL-15Ralpha fragment-Fc on the proliferation of Mo7e cells.
  • Figure 4 shows the effect of the second round of designed IL-15 mutant-Fc/IL-15Ralpha fragment-Fc on CTLL-2 cell proliferation.
  • Figure 5 shows the effects of different IL-15 mutants-Fc/IL-15Ralpha fragment-Fc on NK cell proliferation.
  • Figure 6 shows the effects of different IL-15 mutants-Fc/IL-15Ralpha fragment-Fc on CD4+ T cell proliferation.
  • Figure 7 shows the effects of different IL-15 mutants-Fc/IL-15Ralpha fragment-Fc on CD8+ T cell proliferation.
  • Figure 8 shows the stimulating effect of different IL-15 mutants-Fc/IL-15Ralpha fragment-Fc on NK cell killing.
  • Figure 9 shows the efficacy of HL-015-9 in tumor-bearing mouse models.
  • IL-15/ Dimeric protein form of IL-15Ra Different IL-15/ Dimeric protein form of IL-15Ra.
  • the signal peptide of mouse IL-2 is added (the amino acid sequence of the signal peptide is shown in SEQ ID No. 39, and its corresponding nucleotide sequence is shown in SEQ ID No. 40) and restriction enzyme cleavage sites, and different mammalian cell secretion expression plasmids of IL-15/IL-15Ra dimer proteins were constructed by GenScript Biotech through conventional gene synthesis and molecular construction technologies.
  • heterodimeric protein HL-015-2 as an example to illustrate the construction process of recombinant expression plasmid in eukaryotic cells:
  • the signal peptide with the nucleotide sequence SEQ ID No. 40 is added upstream of the DNA molecule with the nucleotide sequence SEQ ID No. 28 to obtain a new DNA molecule named 28'.
  • the recombinant expression plasmid expresses the protein whose amino acid sequence is SEQ ID No. 12 and the protein whose amino acid sequence is SEQ ID No. 19. The two proteins form the dimer HL-015-2.
  • the knob amino acid sequence information of the Fc fragment is shown in SEQ ID No. 9, and its core
  • the nucleotide sequence is SEQ ID No.37
  • the amino acid sequence information of hole is shown in SEQ ID No.10
  • its nucleotide sequence is SEQ ID No.38
  • the amino acid sequence of the heterodimeric protein HL-015-1 is SEQ No.11 and SEQ ID No.20
  • their nucleotide sequences are SEQ ID No.27 and SEQ ID No.36 respectively
  • the amino acid sequence of heterodimeric protein HL-015-2 is SEQ ID No.12 and SEQ ID No.
  • amino acid sequences of the heterodimeric protein HL-015-3 are SEQ ID No. 12 and SEQ ID No. 20, whose nucleotide sequences are SEQ ID No. 28 and SEQ ID No. 36 respectively.
  • the construction process of recombinant expression plasmids of HL-015-1 and HL-015-3 is the same as that of HL-015-2. The only difference lies in the corresponding replacement of the target coding gene.
  • ALT-803 molecule is named ALT-803 in the patent and is a dimer composed of proteins with amino acid sequences SEQ ID No.3 and SED ID No.4 body);
  • HR-1 the amino acid sequence is SEQ ID No. 5 and SEQ A dimer composed of the protein of ID No. 6;
  • HR-2 the amino acid sequence is SEQ ID No.7SEQ ID No.8 protein composed of dimers
  • the sequence of IL-15 in HL-015-1 (digits 1-114 of SEQ ID No. 11) is consistent with the sequence of IL-15 in HR-2 (digits 1-114 of SEQ ID No. 7) ;
  • the sequence of IL-15R ⁇ subunit in HL-015-1 (No. 1-78 of SEQ ID No. 20) is better than the sequence of IL-15R ⁇ in HR-2 (No. 1-74 of SEQ ID No. 8).
  • the IL-15R ⁇ in HL-015-2 has 13 more amino acids in the sushi region (positions 1-65 in SEQ ID No. 6) than the IL-15R ⁇ in ATL-803 (sequence: DPALVHQRPAPPS).
  • IL-15 in HL-015-3 adopts the N72D mutation
  • HL-015-2 and HL-15-03 HL-015-3
  • the recombinant expression plasmids pCGS3-015-1, pCGS3-015-2, pCGS3-015-3, pCGS3-ALT-803, and pCGS3-HR in Example 1 were transfected respectively.
  • -1 and pCGS3-HR-2 each recombinant expression plasmid was transfected into 200 mL, the plasmid dosage was 0.7 ⁇ g/mL, and the culture time was 10 days.
  • the cell density increased significantly in the early stages of culture, then slowly decreased, and the cell viability was relatively high. When samples were collected on the 10th day, the cell viability was still 80%.
  • Recombinant expression plasmid pCGS3-015-3 Recombinant expression plasmid pCGS3-015-3
  • ALT-803 Expressed by recombinant expression plasmid pCGS3-ALT-803
  • HR-1 Expressed by recombinant expression plasmid pCGS3-HR-1
  • HR-2 Expressed by recombinant expression plasmid pCGS3-ALT-803 Plasmid pCGS3-HR-2 expression).
  • Example 3 Heterodimeric protein affects the proliferation activity of CTLL-2 and Mo7e cells.
  • Cytokine growth-dependent CTLL2 cells (Suzhou Dingding Biopharmaceutical Co., Ltd., Cat. No. TCM-C724) were cultured in RPMI 1640 medium supplemented with 200 U/ml IL-2 and 10% fetal calf serum at 37°C and 5.0% CO2. At logarithmic phase, cells were harvested by centrifugation at 1000 rpm for 5 min and washed three times with phosphate-buffered saline.
  • the heterodimeric proteins HL-015-1, HL-015-2, HL-015-3, ALT-803, HR-1 and HR-2 of Example 2 were respectively serially diluted to obtain heterodimeric proteins. solution, add 10 ⁇ l of heterodimeric protein solution to each well. Each heterodimeric protein is treated with 11 concentrations.
  • the concentrations of heterodimeric proteins in these 11 treatment wells are 500ng/ml, 166.67ng/ml, 55.56ng/ml, and 18.52ng/ml respectively. , 6.17ng/ml, 2.06ng/ml, 0.69ng/ml, 0.23ng/ml, 0.076ng/ml, 0.025ng/ml, 0.0085ng/ml, and set a negative control adding 10 ⁇ l PBS to each well (i.e. 0ng/ ml), set multiple wells for each concentration, and after culturing for another 2 days, add CCK8, and culture for 2 days at 37°C and 5.0% CO 2 Hour. The plate was then read at 450nm and 630nm to check cell growth, and the experiment was repeated three times.
  • the test method for IL-15/IL-15Ra dimer protein to stimulate the proliferation of Mo7e cells is similar to that of CTLL-2.
  • Mo7e cells were added with 8ng/ml GM-CSF and Culture in RPMI 1640 medium with 10% fetal bovine serum to logarithmic phase. Cells were collected and washed, then resuspended in RPMI 1640 medium without GM-CSF and added to a 96-well plate at 20,000 cells per well.
  • heterodimeric proteins HL-015-1, HL-015-2, HL-015-3, ALT-803, HR-1 and HR-2 of Example 2 were serially diluted with PBS respectively.
  • a heterodimer protein solution was obtained, and 10 ⁇ l of heterodimer protein solution was added to each well.
  • Each heterodimeric protein is treated with 8 concentrations.
  • the concentrations of heterodimeric proteins in these 8 treatment wells are 100ng/ml, 33.33ng/ml, 11.11ng/ml, and 3.70ng/ml respectively. , 1.23ng/ml, 0.41ng/ml, 0.046ng/ml, 0.015ng/ml, and set a negative control (i.e.
  • HL-015-2 had the best cell proliferation ability whether it stimulated CTLL-2 or Mo7e (the results are shown in Figures 1 and 2, and the logarithmic values in Figures 1 and 2 are based on 10). HL-015-1 is superior to HR-2.
  • IL-15R ⁇ subunit in HL-015-1 has 4 more amino acids than the IL-15R ⁇ in HR-2.
  • IL-15R ⁇ in HL-015-02 also has 13 more amino acids (DPALVHQRPAPPS) in the sushi region (positions 1-65 of SEQ ID No. 6) than IL-15R ⁇ in ATL-803.
  • Example 2 Based on the experimental results of Example 2 and Example 3, it was decided to select the IL-15N72D mutation shown in SEQ ID No. 3 and the IL-15R ⁇ -Fc shown in SEQ ID No. 19 to continue the following experiments.
  • the synthetic nucleotide sequence is a DNA molecule of SEQ ID No. 34 and the nucleotide sequence is a DNA molecule of SEQ ID No. 35. Add the signal peptide with the nucleotide sequence SEQ ID No.40 upstream of the DNA molecule with the nucleotide sequence SEQ ID No.34 to obtain a new DNA molecule named 34’.
  • the recombinant expression plasmid pCGS3-015-9 contains the HL-015-9 gene (the DNA molecule whose nucleotide sequence is SEQ ID No.34 and the DNA molecule whose nucleotide sequence is SEQ ID No.35), and the expressed amino acid sequence is SEQ
  • the protein and amino acid sequence of ID No. 18 is the protein of SEQ ID No. 19, and the two proteins form the heterodimeric protein HL-015-9.
  • the construction method of recombinant expression plasmids from HL-015-4 to HL-015-8 is the same as HL-015-9. The only difference is that the HL-015-9 gene is replaced accordingly to obtain the HL-015-4 gene (
  • the recombinant expression plasmids pCGS3-HL-015-4 and HL-015-5 genes (the nucleotide sequences are SEQ ID No. 29 and SEQ ID No. 35 respectively) (the nucleotide sequences are SEQ ID No. 30 and SEQ ID No. 35 respectively) ID No. 35)
  • recombinant expression plasmid pCGS3-HL-015-5, HL-015-6 gene (nucleotide sequences are SEQ ID No.
  • recombinant expression plasmid pCGS3-HL- 015-6, HL-015-7 genes nucleotide sequences are SEQ ID No. 32 and SEQ ID No. 35 respectively
  • recombinant expression plasmids pCGS3-HL-015-7, HL-015-8 genes nucleoside The recombinant expression plasmid pCGS3-HL-015-8 (the acid sequences are SEQ ID No. 33 and SEQ ID No. 35) respectively.
  • pCGS3-HL-015-4 table The protein whose amino acid sequence is SEQ ID No. 13 and the protein whose amino acid sequence is SEQ ID No. 19 constitute the heterodimeric protein HL-015-4.
  • pCGS3-HL-015-5 expresses a protein whose amino acid sequence is SEQ ID No. 14 and a protein whose amino acid sequence is SEQ ID No. 19.
  • the two proteins form the heterodimeric protein HL-015-5.
  • pCGS3-HL-015-6 expresses a protein whose amino acid sequence is SEQ ID No. 15 and a protein whose amino acid sequence is SEQ ID No. 19.
  • the two proteins form the heterodimeric protein HL-015-6.
  • pCGS3-HL-015-7 expresses a protein whose amino acid sequence is SEQ ID No. 16 and a protein whose amino acid sequence is SEQ ID No. 19.
  • the two proteins form the heterodimeric protein HL-015-7.
  • pCGS3-HL-015-8 expresses a protein whose amino acid sequence is SEQ ID No. 17 and a protein whose amino acid sequence is SEQ ID No. 19.
  • the two proteins form the heterodimeric protein HL-015-8.
  • the recombinant expression plasmids pCGS3-HL-015-4 to pCGS3-HL-015-9 were expressed and protein purified according to the method in Example 2. Then the activity was measured according to the method of Example 3. The results are shown in Figure 3 and Figure 4 (the logarithmic values in Figure 3 and Figure 4 are based on 10). Among all mutants, HL-015-9 is more effective in stimulating CTLL-2 Proliferation or Mo7e has the strongest proliferative activity.
  • the heterodimeric proteins HL-015-4, HL-015-5, HL-015-6, and HL-015 were serially diluted with PBS according to the different types of dimer proteins.
  • -7. Obtain heterodimeric protein solution from HL-015-8 and HL-015-9, and add 10 ⁇ l of heterodimeric protein solution to each well. Each heterodimeric protein is treated with 8 concentrations. The concentrations of heterodimeric proteins in these 8 treatment wells are 166.67ng/ml, 55.56ng/ml, 18.52ng/ml, and 6.17ng/ml respectively.
  • heterodimeric proteins HL-015-4, HL-015-5, HL-015-6, and HL-015-7 were serially diluted with PBS according to the different types of dimer proteins. , HL-015-8 and HL-015-9 to obtain a heterodimeric protein solution, and add 10 ⁇ l of heterodimeric protein solution to each well. Each heterodimeric protein is treated with 8 concentrations. The concentrations of heterodimeric proteins in these 8 treatment wells are 100ng/ml, 33.33ng/ml, 11.11ng/ml, and 3.70ng/ml respectively.
  • Example 3 1.23ng/ml, 0.41ng/ml, 0.046ng/ml, 0.015ng/ml, the experiment is repeated three times, and a negative control (i.e. 0ng/ml) of 10 ⁇ l PBS is added to each well, and duplicate holes are set for each concentration. .
  • the remaining operations are the same as in Example 3.
  • Example 5 Heterogeneous dimer protein stimulates the proliferation of NK cells, CD8+T cells, and CD4+T cells.
  • NK cells CD56+, CD3-
  • CD8+ T cells CD3+, CD8+
  • CD4+ T cells CD3+, CD8+
  • isolation kits to isolate NK cells, CD8+ T cells, and CD4+ T cells from human peripheral blood cells, respectively.
  • cell The isolated cells were cultured in RPMI 1640 medium containing 10% fetal calf serum at 37°C and 5.0% CO2 overnight, and the cells were collected and stained with carboxyfluorescein diacetate succinimide ester (CFSE) at 37 Incubate at °C for 15 minutes, then centrifuge and wash, and resuspend in RPMI1640 medium containing 10% fetal calf serum.
  • CFSE carboxyfluorescein diacetate succinimide ester
  • HL-015-9 has the strongest stimulation activity of NK cells, CD4+T cells and CD8+T cells.
  • the experiment was divided into 3 groups according to different types of dimeric proteins.
  • concentrations of dimeric proteins ALT-803, HL-015-6 and HL-015-9 were 50ng respectively.
  • /ml, 12.5ng/ml, 3.13ng/ml, 0.78ng/ml, 0.20ng/ml, 0.049ng/ml, 0.012ng/ml, 0.031ng/ml the experimental setting was repeated three times. And set up a negative control (i.e. 0ng/ml) with 10 ⁇ l PBS added to each well, and set up duplicate holes for each concentration.
  • the experiment was divided into three groups according to the different types of dimeric proteins.
  • concentrations of dimeric proteins ALT-803, HL-015-6 and HL-015-9 were 50ng/ ml, 12.5ng/ml, 3.13ng/ml, 0.78ng/ml, 0.20ng/ml, 0.049ng/ml, 0.012ng/ml, 0.031ng/ml, 0ng/ml
  • the experimental setting was repeated three times. And set up a negative control (i.e. 0ng/ml) with 10 ⁇ l PBS added to each well, and set up duplicate holes for each concentration.
  • the experiment was divided into 3 groups according to different types of dimeric proteins.
  • concentrations of dimeric proteins ALT-803, HL-015-6 and HL-015-9 were 50ng respectively.
  • /nl 12.5ng/ml, 3.13ng/ml, 0.78ng/ml, 0.20ng/ml, 0.049ng/ml, 0.012ng/ml, 0.031ng/ml, 0ng/ml
  • the experimental setting was repeated 3 times. And set up a negative control with 10 ⁇ l of PBS added to each well (i.e. 0ng/ml), and set up duplicate holes for the negative control.
  • K562 cells (Wuhan Punosai, Cat. No. CL-0130) were cultured in RPMI 1640 medium containing 5% fetal calf serum at 37°C and 5.0% CO for 48 hours, then BATDA dye was added and incubated at 37°C for 25 minutes.
  • NK cells cultured with culture medium alone were used as a control.
  • Killing rate (%) (mean value of the experimental group - mean value of the blank control group) / (mean value of the maximum release group - mean value of the blank control group).
  • Killing rate (%) (mean value of the experimental group - mean value of the blank control group) / (mean value of the maximum release group - mean value of the blank control group).
  • the experimental results are shown in Figure 8.
  • Each IL-15/IL- All 15Ra complexes can significantly improve the killing effect of NK cells on K562, among which HL-015-9 has the best activity.
  • mice 7-9 weeks old, female, weight range 17-23g, purchased from Beijing Vitong Lihua Experimental Animal Technology Co., Ltd., animal batch number 110011211108488925
  • CT26 cells mouse colon cancer cell line, cultured in In RPMI1640 culture medium containing 10% fetal bovine serum (preserved by Crown Branch)
  • a mouse colon cancer subcutaneous transplantation tumor model was established.
  • the tumor volume reaches 60-100 cubic millimeters, the drug will be administered in random groups (the experiment is divided into 7 groups, with 7 tumor-bearing mice in each group).
  • the control group is the buffer group, and each mouse is injected intraperitoneally with buffer at a rate of 10 ⁇ l/g body weight;
  • the first group is ALT-803, 0.05 mg/kg group.
  • Each mouse is intraperitoneally injected with a solution of heterodimeric protein ALT-803 at 10 ⁇ l/g body weight to ensure the dosage of heterodimeric protein ALT-803. is 0.05mg/kg body weight;
  • the second group is ALT-803, 0.15 mg/kg group.
  • Each mouse is injected intraperitoneally with a solution of heterodimeric protein ALT-803 at 10 ⁇ l/g body weight to ensure the dosage of heterodimeric protein ALT-803. is 0.15mg/kg body weight;
  • the third group is ALT-803, 0.6 mg/kg group.
  • Each mouse is intraperitoneally injected with heterodimeric protein ALT-803 solution at 10 ⁇ l/g body weight to ensure the dosage of heterodimeric protein ALT-803. is 0.6mg/kg body weight;
  • the fourth group is HL-015-9, 0.05 mg/kg group.
  • Each mouse is intraperitoneally injected with heterodimeric protein HL-015-9 solution at 10 ⁇ l/g body weight to make heterodimeric protein HL-015
  • the dosage of -9 is 0.05mg/kg body weight;
  • the fifth group is HL-015-9, 0.15 mg/kg group.
  • Each mouse is injected intraperitoneally with a solution of heterodimeric protein HL-015-9 at 10 ⁇ l/g body weight to make the heterodimeric protein HL-015
  • the dosage of -9 is 0.15mg/kg body weight;
  • Group 6 is HL-015-9, 0.6 mg/kg group. Each mouse is intraperitoneally injected with heterodimeric protein HL-015-9 solution at 10 ⁇ l/g body weight to make heterodimeric protein HL-015 The dosage of -9 is 0.6 mg/kg body weight.
  • the invention provides an IL-15 mutant-Fc/IL-15R ⁇ subunit-Fc heterodimer protein.
  • the heterodimer protein includes protein a and protein b.
  • the protein a can be an amino acid.
  • the sequence is a protein whose amino acid sequence is SEQ ID No. 18, and the protein b can be a protein whose amino acid sequence is SEQ ID No. 19.
  • the IL-15 mutant-Fc/IL-15R ⁇ subunit-Fc heterodimer protein and related biological materials provided by the invention can effectively stimulate the proliferation of NK cells and T cells.
  • dimerization Body protein HL-015-9 inhibits tumor growth better than ALT-803 at the same injection dose. Therefore, the IL-15 mutant-Fc/IL-15R ⁇ subunit-Fc heterodimer protein provided by the present invention can be used to prepare drugs or preparations for treating tumors and/or viral infections.

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Abstract

La présente invention concerne un hétérodimère Fc/IL-15Rα de sous unité Fc portant la mutation IL-15 et son utilisation. La protéine hétérodimère comprend une protéine a et une protéine b, la protéine a comprenant un mutant d'IL-15 et un premier mutant Fc ; la protéine b comprenant un domaine sushi d'une sous-unité d'IL-15Rα et des fragments d'autres parties de la sous-unité d'IL-15Rα, et un second mutant Fc ; et le premier mutant Fc et le second mutant Fc étant choisis parmi un Fc modifié par Knob ou un Fc modifié par Hole, et les types de modification des deux étant différents. Il est prouvé par des expériences que la protéine hétérodimère fournie par la présente invention peut stimuler efficacement la prolifération de cellules NK et de cellules T. Des expériences in vivo montrent que la protéine dimère HL-015-9 inhibe mieux la croissance de tumeurs que ALT-803 à la même dose d'injection, et peut être utilisée dans la préparation de médicaments ou de préparations pour le traitement de maladies telles que des tumeurs et/ou des infections virales.
PCT/CN2023/110205 2022-08-15 2023-07-31 HÉTÉRODIMÈRE FC/IL-15Rα DE SOUS UNITÉ FC PORTANT LA MUTATION IL-15 ET SON UTILISATION WO2024037322A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104093841A (zh) * 2011-06-24 2014-10-08 西图恩医药Sas 基于IL-15和IL-15Rα SUSHI结构域的免疫细胞因子
WO2015103928A1 (fr) * 2014-01-08 2015-07-16 上海恒瑞医药有限公司 Protéine dimérique hétérogène il-15 et ses utilisations
WO2020146835A1 (fr) * 2019-01-11 2020-07-16 Memorial Sloan Kettering Cancer Center Multimérisation de complexes il-15/il-15r-alpha-fc pour améliorer une immunothérapie
WO2021054867A1 (fr) * 2019-09-19 2021-03-25 Закрытое Акционерное Общество "Биокад" Immunocytokine comprenant un complexe protéinique hétéro-dimère à base de il-15 et il-15rα
WO2021119429A1 (fr) * 2019-12-13 2021-06-17 Cugene Inc. Nouvelles protéines de fusion d'interleukine 15 (il-15) et utilisations de celles-ci
CN114341189A (zh) * 2019-06-12 2022-04-12 奥美药业有限公司 全新il-15前药及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104093841A (zh) * 2011-06-24 2014-10-08 西图恩医药Sas 基于IL-15和IL-15Rα SUSHI结构域的免疫细胞因子
WO2015103928A1 (fr) * 2014-01-08 2015-07-16 上海恒瑞医药有限公司 Protéine dimérique hétérogène il-15 et ses utilisations
WO2020146835A1 (fr) * 2019-01-11 2020-07-16 Memorial Sloan Kettering Cancer Center Multimérisation de complexes il-15/il-15r-alpha-fc pour améliorer une immunothérapie
CN114341189A (zh) * 2019-06-12 2022-04-12 奥美药业有限公司 全新il-15前药及其应用
WO2021054867A1 (fr) * 2019-09-19 2021-03-25 Закрытое Акционерное Общество "Биокад" Immunocytokine comprenant un complexe protéinique hétéro-dimère à base de il-15 et il-15rα
WO2021119429A1 (fr) * 2019-12-13 2021-06-17 Cugene Inc. Nouvelles protéines de fusion d'interleukine 15 (il-15) et utilisations de celles-ci

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